There currently exist methods that can affect the rates by which biological metabolic processes proceed. The ability to alter these processes would find applications where reductions in biomass and biofilm production, accelerated nutrient uptake, improved fermentation rates, as well as methods for altering biochemical processes would be beneficial.
Acceleration of nutrient uptake without a concomitant increase of biomass can be achieved by uncoupling biochemical degradation (catabolism) from biochemical synthesis (anabolism). Uncoupling can occur during oxidative phosphorylation resulting in lower adenosine triphosphate (ATP) formation, or by dissipating generated ATP through “energy spilling”. There are some chemical moieties known to uncouple oxidative phosphorylation, however they are inherently toxic and cost prohibitive.
All microorganisms have the common purpose of using catabolism to conserve free energy by distributing it among compounds that can store and carry energy to where it is required in the cell. Intracellular regulation of catabolic and anabolic processes by bacteria is necessary to ensure an efficient flow of energy. Atkinson B. and Mavituna F., Biochemical Engineering and Biotechnology Handbook, 2nd Edition, pp. 130-131, Stockton, New York (1991), describe the role of adenosine diphosphate-adenosine triphosphate (ADP-ATP) as follows: The high-energy phosphate bonds of ATP are used in coupled reactions for carrying out energy-required functions, wherein ultimately ADP and inorganic phosphate are formed. ADP is rephosphorylated to ATP during energy yielding reactions of catabolism. (See FIG. 1). Within the mitochondria of higher organisms, the concentration of ATP is known to regulate the activity in the citric acid cycle, in effect producing a feedback control loop. (Stryer, L., Biochemistry, 3rd Edition. Freeman, New York (1988)).
Senez, J. C., Some Considerations on the Energetics of Bacterial Growth, Bacteriol. Rev. 26, 95-107 (1962), suggests that bacterial anabolism is coupled to catabolism of substrate through rate-limiting respiration. However, uncoupled metabolism would occur if respiratory control did not exist and instead, the biosynthetic processes were rate limiting. Therefore, the excess free energy would be directed away from the production of biomass.
Senez, in describing the link between energy-yielding reactions and the energy-consuming reaction of cell biosynthesis, conceptualized any anomaly as “uncoupling”. Russell and Cook (Microbiological Reviews, March 1995, pp. 48-62) declared this all-inclusive definition did not differentiate between the production of ATP and the utilization of ATP in non-growth reactions. Because the latter process would be more aptly termed ATP energy spilling, therefore defining uncoupling as the inability of chemiosmonic mechanisms to generate the theoretical amount of metabolic energy in the form of ATP. This is redefined as “uncoupled oxidative phosphorylation” to differentiate it from other mechanisms.
Stouthamer, A. H., Correlation of Growth Yields In Microbial Biochemistry, International Review of Biochemistry, Ed. J. R. Quayle, Vol. 21, pp. 1-47, University Park, Baltimore (1979), reports that uncoupled metabolism has been observed in the presence of the following conditions: 1) in the presence of inhibitory compounds, 2) in the presence of excess energy source, 3) at unfavorable temperatures, 4) in minimal media, and 5) during transition periods in which cells are adjusting to changes in their environment.
Low and Chase (Wat. Res., Vol. 33, No. 5, pp. 1119-1132, 1999) theorize that decreasing the ATP available for biosynthesis would, in turn, reduce biomass production. The ability to replicate these uncoupling processes in wastewater treatment would be advantageous. Further, if microorganisms exhibit similar behavior to mitochondria in the regulation of the activity in the citric acid cycle, then a reduction of cellular ATP production would provide a stimulus to the feedback loop to promote accelerated catabolism of pollutants.
There exist several possibilities for the consumption or loss of energy required for biomass production, including the dissipation of energy as heat by adenosine triphosphate systems, the activation of alternative metabolic pathways bypassing free energy conserving reactions, and the accumulation of polymerized products in storage form or as secreted waste.
Protonophores are reagents that exhibit the ability to disrupt the tight coupling between electron transport and the ATP synthase of the respiratory chain, because they dissipate the proton gradient across the inner mitochondrial membrane created by the electron transport system. Typical examples include: 2,4-dinitrophenol, dicumarol, and carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone. These compounds share two common features: hydrophobic character and a dissociable proton. As uncouplers, they function by carrying protons across their inner membrane. Their tendency is to acquire protons on the cytosolic surface of the membrane (where the proton concentration is high) and carry them to the matrix side, thereby destroying the proton gradient that couples electron transport and the ATP synthase. The energy released in electron transport is dissipated as heat. Biochemistry, 2nd Edition, Garrett and Grisham (1999)
Addition of protonphores to uncouple the energy generating mechanisms of oxidative phosphorylation will stimulate the specific substrate rate uptake while reducing the rate of biomass production.
Low and Chase (Wat. Sci. Tech., Vol. 37, No 4-5, pp. 399-402, 1998) supplemented a chemostat monoculture of P. putida with the protonphoric uncoupler of oxidative phosphorylation, para-nitrophenol. The effect of this addition was to dissipate energy within the cells and thus reduce the energy available for endothermic processes. Under these conditions, cells continued to satisfy their maintenance energy requirements prior to making energy available for anabolism, thus reducing the observed biomass yield.
Through catabolism, cells make biologically useful energy available for fueling their endothermic reactions. An increase of the energy requirements for non-growth activities, in particular maintenance functions, would decrease the amount of energy available for biosynthesis of new biomass. Endothermic maintenance functions include the turnover of cell materials and osmotic work to maintain gradients. The net effect is a utilization of ATP through ‘futile cycles’ also called energy spilling, which reduces the amount of ATP available for the synthesis of biomass. Also, energy requirements for cell motility cannot be differentiated from maintenance energy requirements. (Low and Chase, 1998)
The discovery and use of peptides produced from Saccharomyces cerevisiae for medicinal applications described by George Sperti in U.S. Pat. Nos. 2,320,478, 2,320,479 and 2,239,345, illustrate a method for producing low molecular weight heat-shock proteins, referred to as Live Yeast Cell Derivative (LYCD), that has demonstrated the ability to affect skin respiration. The active peptides in the LYCD have been isolated and identified by a number of individuals. Bentley, U.S. Pat. No. 5,356,874 and Bentley et al. (Peptides From Live Yeast Cell Derivative Stimulate Wound Healing, Arch Surg, Vol. 125, pp. 641-46, May 1990) describe the active ingredients as an angiogenic factor comprising a mixture of polypeptides having molecular weights ranging between about 6,000 daltons to about 17,000 daltons, said factor isolated from a yeast cell derivative. Schlemm et al. (Medicinal Yeast Extracts, Cell Stress & Chaperones (1999) 4(3) 171-76) further defines these extracts as the 15,700 dalton yeast copper, zinc-superoxide dismutase, the 10,100 dalton acyl CoA binding protein (ACBP), the 8,560 dalton ubiquitin protein and the 7,090 dalton peptide of the C-terminal fragment of heat-shock protein 12, a glucose-lipid regulated protein.
Enzymatic compositions founded on yeast extracts have been used for the treatment of wastewater. Battistoni, U.S. Pat. No. 3,635,797, describes a multi-enzymatic composition comprised of an enzymatic fermentation reaction product, surfactants, citric and lactic acids, urea and pine oil. Battistoni claims the composition greatly improves sewage treatment facility capabilities by stimulating bacterial growth, eliminating odors, and enzymatically improving the catalytic degradation of sewage impurities. The Battistoni patent describes an anaerobic yeast fermentation process as follows:
“Approximately 1,000 gallons of warm softened water having a temperature of between about 85-100 degrees F. was placed in a large tank. To the water was added 700 pounds of black untreated cane molasses, 210 pounds raw cane sugar and 10 pounds magnesium sulfate. The mixture was thoroughly blended, after which 95 pounds diastatic malt and 10 pounds bakers yeast were added and agitated slightly. The composition was allowed to stand for about 3 days, after which the effervescent reaction had subsided, indicating essentially complete fermentation.”
Because it is performed without active aeration or agitation, the fermentation process described in Battistoni is considered anaerobic in nature.
Dale, U.S. Pat. No. 5,879,928, describes a composition for the treatment of municipal and industrial wastewater, comprised of a yeast fermentation supernatant, preservatives and a non-ionic surfactant. The composition comprises a fermentation supernatant from a Saccharomyces cerevisiae culture, sodium benzoate, imidazolidinyl urea, diazoldinyl urea and a non-ionic surfactant. The Dale patent describes a composition having desirable properties associated with surfactant micro bubbles. Dale explains that the micro bubbles formed with the composition appear to increase the mass transfer of oxygen in liquids. Further, the micro bubbles are the result of aggregates of surfactant molecules with a loose molecular packing more favorable to gas mass transfer characteristics because a surface consisting of fewer molecules would be more gas permeable than a well-organized micelle containing gas. Dale further describes biologically derived catalysts in combination with the surfactants, both of which tend to be amphiphilic; that is, they have pronounced hydrophobic and hydrophilic properties. The non-ionic surfactants used in the Dale composition are said to be compatible with, and enhance enzymatic reactions. However, Dale also states the composition has catalytic activities that are more like the catalytic activities of functionalized surfactants than conventional enzyme systems.
The composition of the Dale patent is similar to that described in the Battistoni patent; that is, the fermentation process is anaerobic in nature, with an added step of the removal of the resulting yeast cells by centrifugation. The Dale fermentation process is described as follows:
“The yeast, Saccharomyces cerevisiae, is cultured in a medium comprising: a sugar source, such as sucrose from molasses, raw sugar, soy beans or mixtures thereof. A sugar concentration of about 10 to about 30%, by weight; malt, such as diastatic malt at a concentration of about 7 to about 12%, by weight; a salt, such as magnesium salts, and in particular magnesium sulfate, at a concentration of about 1 to about 3%, by weight, and yeast is added to the medium to a final concentration on about 1 to about 5%, by weight, is used. The mixture is incubated at about 26 degrees to about 42 degrees C. until the fermentation is completed, i.e. until the effervescence of the mixture has ceased, usually about 2 to about 5 days, depending on the fermentation temperature. At the end of the fermentation, the yeast fermentation composition is centrifuged to remove the “sludge” formed during the fermentation.”
Consistent with the Battistoni patent, without the presence of active aeration or agitation, the fermentation process described in the Dale patent is considered anaerobic in nature. Carbon mass balance studies, conducted under controlled conditions, indicate the Battistoni composition increases the rate of carbon metabolism versus an untreated control. Likewise, carbon mass balance studies conducted with the Dale composition yielded an even greater increase in the rate of carbon metabolized versus Battistoni and the control. However, the rate of conversion of carbon metabolized to biomass carbon of either composition remained relatively consistent with the conversion rate of the untreated control. This would indicate the uncoupling of metabolism is not a function of either the Dale or Battistoni compositions. Nor are the low molecular weight peptides described in the present application produced as a result of the anaerobic fermentation of Saccharomyces cerevisiae as described in either of the Battistoni or Dale patents.
Production of excess biomass during biological treatment of wastewaters requires costly disposal. With environmental and legislative constraints limiting disposal options, considerable impetus exists for reducing the amount of biomass produced. (Low E. W. and Chase H. A., The Effect of Maintenance Energy Requirements on Biomass Production During Wastewater Treatment, Water Research, Vol. 33, Issue 3, pp. 847-853. (2000)). The activated sludge process employs a microbial population that will convert organic pollutants to cell mass and respiration products. Cell mass accumulates within the process and the excess biomass must therefore be disposed of. Although such treatment and disposal may already account for 60% of total plant operating costs, (Horan N. J., Biological Wastewater Treatment Systems, Wiley, Chichester (1990)), these costs are expected to rise with new European Community (EC) legislation and decreasing landfill availability. Sludge disposal in the United States has also come under increasing scrutiny and new legislation regulating sludge disposal is being enacted by government agencies at all levels. (Low and Chase, The Use of Chemical Uncouplers for Reducing Biomass Production During Biodegradation, Wat. Sci. Tech., Vol. 37, No. 4-5, pp. 399-402, 1998).
Dissipating energy intended for anabolism of cell mass without reducing the rate of removal of organics from aqueous waste provides a direct mechanism for reducing the yield of biomass. The chemiosmonic mechanism of oxidative phosphorylation (by which adenosine diphosphate (ADP) to energy-rich adenosine triphosphate (ATP) is produced during catabolism, (Mitchell, P., Chemiosmonic Coupling and Energy Transduction: A Logical Development of Biochemical Knowledge, Bioenergetics 3, 5-24 (1961)), can be uncoupled using protonphores and under these circumstances is dissipated. Oxidation of the substrate still occurs, but the phosphorylation of ADP to ATP is reduced, and consequently, there is less energy available for the formation of biomass. (Simon, E. W., Mechanisms of Dinitrophenol Toxicity, Biol. Rev. 28, 453-479 (1953)).